1. Field of the Invention
The present invention relates generally to the field of cell adhesion molecules and specifically to the use of oligosaccharide compositions for modulating selectin receptor binding.
2. Description of Related Art
Cell adhesion molecules (CAMs) play a role in inflammation, infection, cancer and other disease processes. Recent advances in cloning and protein sequencing have led to the organization of CAMs into families, based on their molecular structure. Intense research has been focused on selectins, which are carbohydrate-binding proteins expressed on endothelial and leukocyte surfaces; B2 integrins, which are heterodimeric proteins found on the surfaces of leukocytes; and proteins of the immunoglobulin type such as intracellular adhesion molecule, ICAM-1 and ICAM-2 which occur on many different cell surfaces.
The mechanism of inflammation can be understood in terms of CAM interactions. Leukocyte adhesion to the vessel wall is a key step in the development of inflammatory and immunological processes. Adhesion molecules that support these interactions include the selectins, a group of CAMs which are named for the cell type on which they were originally identified. The selectins include E-selectin (endothelial cells), P-selectin (platelets and endothelial cells) and L-selectin (lymphocytes).
The three selectins act in concert with other cell adhesion molecules (e.g., ICAM-1, vascular cell adhesion molecule-1 and the leukocyte integrins) to effect adhesive interactions of leukocytes, platelets and endothelial cells. E-selectin was first shown to support the adhesion of neutrophils to cytokine-activated endothelium (Bevilacqua, et al., Proc. Natl. Acad. Sci., USA. 84:9238, 1987; Bevilacqua, et al., Science 243:1160, 1989). Subsequent studies in vitro have suggested that E-selectin also supports the binding of monocytes, a sub-population of memory T lymphocytes, eosinophils and basophils. Similarly, P-selectin also supports leukocyte adhesion. In addition to its role in lymphocyte homing, L-selectin appears to participate in the adhesion of neutrophils, monocytes and lymphocytes to activated endothelium (reviewed in Bevilacqua, M. and Nelson, R., J. Clin. Invest. 91:379, 1993).
Selectins contain domains homologous to C-type lectins, therefore there has been an intensive search for carbohydrate ligands. Many recent studies on selectin-carbohydrate interactions have focused on oligosaccharides. P- selectin-dependent rosetting of activated platelets to leukocytes is blocked by LNF-III, a pentasaccharide containing the Lewis x determinant (Le.sup.x ; Gal.beta.1-4(Fuc.alpha.1-3)GIcNAc) (Larsen, et al., Cell, 63:467, 1990). Other studies identified the sylated form of this oligosaccharide, sLe.sup.x (Neu5Ac.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GIcNAc) and/or closely related structures as ligands of E-selectin. sLe.sup.x and other fucosylated lactosamines are found in abundance on circulating neutrophils and monocytes and on a small percentage of blood lymphocytes (reviewed in Bevilacqua, M. and Nelson, R., supra). Separate studies have demonstrated that sialic acid is a component of some P-selectin ligands, and that oligosaccharides containing sLe.sup.x are recognized by this molecule. In addition, human E- and P-selectin and murine L-selectin have been shown to interact with molecules containing sLe.sup.a (Neu5Ac.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GIcNAc), a structural isomer of sLe.sup.x. sLe.sup.a is not typically expressed by blood leukocytes, but is expressed by certain cancer cells, suggesting a possible role in metastasis.
In response to certain mediators like thrombin and histamine, endothelial cells redistribute P-selectin from storage granules to the surface within minutes. In response to endotoxin, IL-1, or TNF, endothelial cells biosynthesize and express E-selectin as well as VCAM-1 and ICAM-1 over a period of hours to days. L-selectin is constitutively expressed by leukocytes and appears to recognize a cytokine-induced endothelial cell surface ligand. Inflammatory processes are essential for host defense against pathogens. When control mechanisms fail or the pathogen burden is too great, the inflammation becomes extreme. The ensuing tissue damage contributes to important human diseases such as rheumatoid arthritis, ischemic reperfusion injury, autoimmune diseases, and adult respiratory distress syndrome (ARDS).
Heparins are widely used therapeutically to prevent and treat venous thrombosis. Apart from interactions with plasma components such as antithrombin III or heparin cofactor II, interactions with blood and vascular wall cells may underlie their therapeutic action. The term heparin encompasses to a family of unbranched polysaccharide species consisting of alternating 1.fwdarw.4 linked residues of uronic acid (L-iduronic or D-glucuronic) and D-glucosamine. Crude heparin fractions commonly prepared from bovine and porcine sources are heterogeneous in size (5,000-40,000 daltons), monosaccharide sequence, sulfate position, and anticoagulant activity. Mammalian heparin is synthesized by connective tissue mast cells and stored in granules that can be released to the extracellular space following activation of these cells. Overall, heparin is less abundant than related sulfated polysaccharides, such as heparan sulfate, dermatan sulfate, and chondroitin sulfate, which are synthesized in nearly all tissues of vertebrates. Heparin and these other structures are commonly referred to as glycosaminoglycans.
The anticoagulant activity of heparin derives primarily from a specific pentasaccharide sequence present in about one third of commercial heparin chains purified from porcine intestinal mucosa. This pentasaccharide, --.alpha.GIcNR.sub.1 6S.beta.(1-4)GIcA.alpha.(1-4)GIcNS3S6R.sub.2 .alpha.(1-4)IdoA2S.alpha.(1-4)GIcNS6S where R.sub.1 =--SO.sub.3 .sup.- or --COCH.sub.3 and R.sub.2 =--H or --SO.sub.3 .sup.-, is a high affinity ligand for the circulating plasma protein, antithrombin (antithrombin III, AT-Ill), and upon binding induces a conformational change that results in significant enhancement of antithrombin's ability to bind and inactivate coagulation factors, thrombin, Xa, IXa, VIIa, XIa and XIIa. For heparin to promote antithrombin's activity against thrombin, it must contain the specifically recognized pentasaccharide and be at least 18 saccharide units in length. This additional length is believed to be necessary in order to bridge antithrombin and thrombin, thereby optimizing their interaction.
Much of the recent work on selectin-carbohydrate interactions has focused on oligosaccharides based on Le.sup.x, Le.sup.a, and sialylated and sulfated derivatives of these structures. While crude fractions of heparin have been reported to bind and inhibit P-selectin-dependent and L-selectin-dependent interactions, no reports have shown studies on binding of small heparin fragments to the selectins. The specificity of heparin binding to the selectins is unclear, although other sulfated polysaccharides such as fucoidan and dextran sulfate also bind to P-selectin and L-selectin.
Crude heparin fractions have been reported to affect the activity of mast cell granule components, including histamine, and have been known to dampen allergic responses (Higginbotham, et al., P.S.E.B.M. 92:493, 1956; Carr, J., Thromb. Res 16:507, 1979). In rat models, heparin causes enhancement of certain platelet responses (Srivastava, et al., Biochem. Pharmacol. 40:357, 1990), reduction of carrageenin-induced footpad edema (Hanahoe, et al., Int. Arch. Allergy Appl. Immunol., 86:243, 1988), and inhibition of allergic encephalomyelitis (Willenborg, et al., J. Immunol. 140:3401, 1988). Inhaled heparin in a sheep model reduced antigen-induced bronchoconstriction (Ahmed, et al., Am. Rev. Respir. Dis., 145:566, 1992). Crude (unfractionated) heparins have also been observed to modulate various immune responses in animal models (Gorski, et al., FASEB J. 5:2287, 1991). These include delayed-type hypersensitivity reactions (DTH), allograft rejection, and immune cell-mediated killing. Tumor metastasis may also be affected by heparin. Most of these studies described above used crude heparin fractions of average molecular weight approximately 12000-15000 (40-50 saccharide residues) which would also possess significant anticoagulant activity.
Thus, considerable need exists for compositions with the biocompatibility and antiinflammatory properties of crude heparin without the danger of systemic anticoagulant activity. The present invention provides such compositions.